PHOTOCHEMICAL FORMATION OF SELF SUSTAINING COACERVATESI By
Knrsnxl
.
Chemistry Department, Allahabad University, Allahabad, INDIA
proble- of otigin of lile started,fl"-i ll"f:i,'.:i9*-' ol Haldane & Opaiin, commonly k1^own as [he Molecular or Chemtcal -F-volu(lon (Haldane I 929, Oparin l938). theorv "''"'tTil;;;;";;l*;"r irrolr.tion has been used in a restrcted sense. I1 olly of ,ooiecrrles which formed the earliest ,cells,-by i".*uiio" ultimate lfr" *.ur., o1 earth (Bahadur 1967)' The ;1.;i";f;r;*.io.*utioos which were taking plage_ where it is believed biological.sense in the strici used not been has evolution term which even on system replicating a hrrre fotlo*, o"iy *h"" *e ifr.i '..url"r Actualiy fotm' "u., m*odifi"d in'this "|f"ii"r, can duplicate t ut"tui Uy modification natural the search is to lile of oiigi" investigation on of the ;h;;;;;rptr. to begin 'vith systems ieplicating"seTf-sustaining *""tt fbrm"a which conditions Bahadur and Ranganayaki 1966)' igtn* ' The1961, is that it is taken second reservation in thE thebry oi *oi.".tiut evolution whicharefound materials t6.*u*" ,,'id"of fo. g.1.1".d ifrli*."*fiest cells *.." nrobable that the is,equally^ it big assumptiol It is a cells. day pr.r"nt ii.',fi" for not have bedn there to begin ..il *ight chemicals which ar.e pr.esent in today's 'of tot".-.""olutionary metabolism operating in with and were formed'a, tfr. p.oa".t (Bahadur 1967). the "'" cells day.cell werc made ;l* !,'11" pt"fr^"tj"'ihu, the_precursors of the present acids in nature was amino naturai of of i"art .=*ioo'u"iar-r"J tf." abiigenesis ^t ,",,Sirl.- Witf, the same pro"l"r, oitoni" thc abiogenesis ol almost all the il*if' thc iast lwo dccades Ihemicals present i" ;;r;^;;;;; i;; ""ttr ** ?eurcled cluiing formation of Photo-ch"emical encouragrng';"'"rit' ,r..y il:'1;i;';i.l;;;'ro*" or nitric ammorriacal iiri""'".rAt i" rr".irir.a mixtures .o,"."tuiXi*g-iormaldehyde, has been observed by catalysts inotlanic as or molybdenum iron and 'nitr'oqen 1e55, Bahadur et' al' 1e5B)' B;h;e;; iil-ur,,a". i'gs,il"ii"r',a*;;J R;"!;;ayaki pavlovskava ur-,a eurf.,ir.ii irssz; obtain;d- u-#no u.idr by exposing similar \-ilffit-;; "il;; ;;fi ruyr) nrJ"y oth"t toui.es of energv'have-been used for amino acid sYnthesis. peptides'of low mol' In 1957 Bahadur observed that on exposure to sunlighta'mino acids',-sug?l-ls -ut containing wt. are-f**.d i" r[tiiir"J ;q".;;, mixtires l95B' (Bahadur c"atalysts inorganic o, *6lybd.,r,'r* it." energv source -rii"-ior,.'rufion been lras mecjium "r,a aqtueous of p.piiJ"t itfilYtritil"igoli. ageni condensing as dicyanamide also,'nJitg rrrJ-sl.i.r-an Ji"ii.i 't\'f"'C;I"i" -l.Presenteilinthe4thlnternationalslmposiumon"OriginofLtftontheEarth"' held at Barcelona, Spain in June 1973' The present approach to
['.
Bane.oun
th-e
i"
-2(steinman and Lemmon 1964, 19G5, steinmar and Kenvon 1965). several detailed reviews on abiogenesis are 19r,v avli]alle (Steinmarr l96g;oparin lg63). . T!. important point irr the origin of life is to investig'ate a* ilo# these mole.
cules formed the earliest cells. The work on abiogenesiJ becomcs pertinent in solving the problem-of life synthesis only if self-sustiining systems are synthesised
usinE these molecules. Backward exploitation of the diversity of the phylology in abiogenic time suggesls tha.t to begin with tlrere nrust have been few ipcciFi (eirie lSS4). T6e rvork of Dillon (1962) shows that the problenr of oligin^of orsdnellcs of i cell is not the problem, of_origin of liG but that of evolutioniry cytoldgy arrd thc earliest cells must have had very simple internal structures. Lwoff's (ili43) work on the Ioss or gain of the properties of individual c-ells during evolution rvith time suggests that the evolution is loss and not gain in the properiie* of the iqdividual celTl . Putting the work of Dillon and Lwoff to$etlier it appeals that to begin witlr there were fewer species and the earliest cellu'iar living systems were vcr! simpte in strttctrrre and werc i'ull or'propcrties of biological orclcr (Bahadur l9'64) a;cl these were.the precursors of ceilulir life. 'the stJdy of origin'of life is the investigation of the natural formation of these precursors of c.llirlar life. Bahadur, matter has inherent property ol duplication under .. ,According.to suitable conditions a.1d a system of matter in equilibrirrm his an inherent Rrgperty of^ adaptability (Bahadur and Ranganayaki 1964, Bahadur 1967, Bahadur 1964). Ihe nucleic acid assisted duplication of protein molecules is common in all the,livings of the present day. liid_livir,g for^ms originate initially u* u p.ot"i* nuclerc acld system or wel'e they made of some simple molecules of low molecular weight and the living forms of high molecular weighf molecules are the evolutionary product of th_e earlier^forms, is an open questionl It appears more probable thai to begin with living forms were -id. o? small molecules and the'nucleic acidPI91"i1 made living forms resulted as the evolutionary product afterwards (Bernal 1961, Bahadur 1964, Bahadur et. al. 1966). If so' ihe smaller moleculis can duplicate Fy ttt" quantum mechanical .esotr.ince interaction special stability force considerations- only (Jordon 1938, tg3g, lg40). The molecules of such 'livine forms could duplicate by themselves wiihout ir.eeding the help or nucleic ac;dl II-any mild change in the duplicating molecule was briught abour by the phvsicochemical conditions of the Cnvironrient, the slightty nioalnea form duplicate and this system if in equilibrium with lts environment could "o,-ild'ulro adapt and such a system .would have been capable of evolution. Though it appears,to be_easy to differentiate a living from a non-living rI*.*: it.is.very difficult to define life in scientific languige. Haldane (lg54j dehned a-hving system as-a self perperuating system. Accordlng to Bernal itgszi the embodiment of the sellperpetuating systein irittt-;n a boundaiy was the ociarioti oF origin of life. Ho'owilz (tssz; d*efined living system as a monomolecular system.in polymolecular environment, capable "of 'multiplication and heterocatalysis. Konikova (1957) suggested-thaf a living system is a molecule or a complex which by the process i-f chemical reactioirs with the molecules of its envlronment accomplishes growth dnd multiplication. ft remains to itself while yet changing not in the direction of death oi decay. Pirie (1957) however did not agree to the limitatiou of growth f,nd multiplication for thl definition of u fi"ing system.
In 1967 the Physicist J. D. Bernal gave a definition -p;;t of life in electronic language. According'to hiri life is a coitinuo.rq *.rtiiform and con-
-3ditionally inter active, self realisation of the potentialities of the atomic electron state.
I
t I
It thus appears that it is very difficult to draw a line of demarkation between livings and non--livings and defining life is a hopeless job. llowever it became necessary to at least enumerate a few properties which a system must have before it can be included i}^t" category of-living systems. Bahadur made an attempt in this direction in 1963. _Accbrding to him if a system is capable of growtir, multiplication and metabolic activitv the system b" included in the "unsize of the systemfromwithin of living, where growth standsfor th6 increaie in the "it.gory by actual synthesis of the material with which the system is rnade, inside the system, rnultiplication means the system increases in number and the nelver units'come in existence through . the parent ones and metabolic activity denotes any series of chemical reactions taking place within a boundary the result of which is that at least a part of the environmental molecules enterinq the systern is converted into the material with which the system is made and fhese chemical transformations
p.rovide the various energy requirements of the system lor performing these functions
1
t'
I I t'
t
(Bahadur and Ranganayaki- 1964, Bahadur IOOT;. About the chemical nature of the living system Pirie (1957) holds that proteins are necessary for the present day cell because_they are enzymss and catalyje many biochemical reactions. But miny metallic ions'also show enzyme like activity and it is possible that to begin with these might have been periorming the worf of protein el?ryngs.- Bernal-(1957) is of the opinion that there might-have been organisms which had only inorganic catalysts in the place of proiein and such organisms might have been slufgish but could have certainly performed all the functions of life. According to-Smirnova (1957) many inorganic substances have physical properties. commonly found in organic iubstances aia it is quite possibie to.conceive organisms made of these inoiganic substances only. Bernal- (196l) writes "unless ft is desired to push back tEe doctrine of speciJl creation io thi, creation of enzymes and co-enzymes (there is a school that wbuld take one of these, n?T:ly the coenzymes irr the polymi:rised form as nucleic acid) as the beginning o_f life, unless then we are prepared to take such an easy way orrt, *c must assume that before there were enzymbs to carry out the catalyiic rea-ctions in metabolism there were some other- agents that did it, not so well,'but sufficiently well for the slow time of the origin-of life." These-evolved to produce protein-nucleic acid cellular life which *."rro* observe on the earth. Bahai.,. hur siggested a proba.fle locale for these forms of life (Bahadur and Ranganayaki 1964, BI-iiadur 1ti64,1967
and Bahadur et. al. 1966). _ . O*9 of the important aspects of the problem of origin- of li{b is to study the ph_i-sico-chemical faitors which brought the molecul"r #hi.h formed the e#liest ce ll together, kept -them held togethei and arranged them in some specific pattern that could show the properties-of biological order. Irr 1963 Bahadur photo-chemically-synthesised a type of particleswhich he narned as-J99wq1u (Bahadur et. al. 1964, Bahadur and dar,.ganayaki 1964, Bahadur 196+, 1967, Bahadur et. al. 1964). In the sterilised aq:.,"n.ts mixture coEtainirrg. or"gauic carbon,_ inorganic nitroger. an-d minera-ls comrnonly foun-d in cells, sma1l spherical particles ari formed rihich have definite boundary wail arrd irrtricate internal struttures (Nlicrograph l). 'Ihese particles are very'similar to the present da;' cell in chem,ical composition and differ from the .odmon microolganisms J!l! tlqy ca.11n-ot be grown on. a.ny knou,'n bacterizrl cu,lture medium (Bah.adur' 19.6+._ 1.966, 1s67, Balldur and Ranganayaki 1970). 'fhese particles multiplr' by budding (Micrograph 2) and the small buds grow to maturity size
-+and bud (Micrograph 3). Bahadur emphasised that in rvater where organic materials and necessary inorganic substances wer-e present, sun light synthesised amino acids, peptides, sugars, and such other biochemicals and ihese organised in the form of microstructures and formed Jeewanu (Micr:ograph l4) . TheseJeewanu into thepresent day cellularlile wcrecapableofadaptalrilityandsotheseevolved (Bahadur et. al. 'l963, Bahadur 1967 1. The work onJee\vanu \\'as soon repeated by Briggs and his confirmations of this work was read in the 4th .Interrrational Sympcsiurn on Photobiologl, held at Oxford in 1964. He further repeated. some experimentsof Bahadur and published another confirmation in Space Flight in 1965. Irr 1970 the rvork onJeewanu was further con-firmed by Nlueller and Rudin. A complete review of the rvork on Jeewanu has also appeared (Bahadur 1966, 1967). B'ahadur and Ranganayaki photochemically produced sell sustainirr.g coacervates by the interaction of ammorrium molybdate, diammonium h,vdrogen phosphate, minerals commonly found in cells and formaldehyde, in aqueous mixtures (1970) (Microeraph 10, 12, 13 and 1t) The experiments are so simple tha.t those can be given as a class exercise for graduate students. . In the Jeewanu the property of growth, multiplication and metabolic activity is observed in a natural way, once the experiments are set and no specific chemicals are needed for any specific property (Bahadur and Ranganayaki 1966). These particles have a number of amino acids in free form aud in combin'd form as peptides, and sugars as ribose, deoxyribose, fructose, and glucose. They have distinct boundary wall and intricate ieternal structure. The internal structures can be clearly viewed under high magnification (Micrograph 4) . Micrograph of the particles under phase contrast microscope reveal the boundary wa.ll and the internal structure of the particles clearly (Micrograph 5 and l1). These particles multiply by buddirrg (Micrographs 6,.7, B, 9 and 10) The prticles on separation frorn the mixture if extracted with chloroform metha.uol : B0 : 20 in a soxlrlet yield a viscous yellow liquid. This contains an ethyl alcohol soluble component which on chromatography gives the test for phospirolipids (Bahadur and Singh 1973). 'Ihese particles on hydrolysis with perchloric acid or formic acid in sealed tube give the tests for nucieic acid bases as. adenif.e, guanine, cytosine, thymine and ulacil. If the particles are kept in 1 N sodium hydroxide for 24 hours, filtered and the filtrate acidified rvitir dilute acetic acid a white precipitate is obtained which on subsequent hydlolysis gives the test of desoryribose nucleic acicl (Bahadur and Ranganayaki 1970, Rarr.ganayaki et. al. .
re72).
The particles can Jle fixed with chlomic acid and subsequently stained lvith gcrrtian violet and then eosirr. In the centrai portion chromatine-like blue structures are obtained and the portion outside the central zone gets red stain with eosin,
like cytoplasm cvtoplasm (Bahadur and Gupta
It
1972\. 1972).
has been reported that the materials of the particles on digestion 'drocFlffi acld show strong optrcal activlty (lJaha.dur and l(anganayaKL l tors which are responsi6le-T6rTlie natural formation of objects a definite morphology have been discussed by Bahadur (1964, 1967). The molecules of differen-t chemicals are brought together and are first held by coacervate forming factors. If this contains some macromolecules which have a number of molecules attached to ii by various intermolecular forces as van der \\raals forces, hydrogen bonding, hydrophobic bonding, molecula.r bonding and others an-d also have many molecules a-bsorbed, adsorbed and held together by electrostatic forces, when this macromolecule tries to crystallise, it forms a highly deformed crysta.l and the rvhole thing results in a molecular mesh having wide
-5-
{hi J i ^'r
gaps and passages through which ^sma|l environnlental molecules- have- specific ,ru.ioi,r gaps .of different molqcules held in this deformed [.i^.u6itiiy. f6"r"tr^ir'u.ti"d chernically and also . catalytica"llv. The whole iryrtut stru'cture en-etgptic patiern representin'g the structure in- an attempt to accluire a spatio ^a stale of minimum .'n..qy .c'.ults in biological looking structute'anThc outcr .intricate material forms a. boundaif wall and the aggre[ate appeari to .have int..nul structure. If thii structure is present in an approp^riate environ'ment source,of ;;;;;i;ilg *or.."f"r *frilf,, ca-rr form its Uoay matelial .and,if .it hasoraevolvcd by enersy, may be ol ,o*. plrysical llatu|e as obtained b)'.ir|adialion the environmental the mi-xture, in ta.kins place ,""*""'Jfr.-i"J"t.i"tfo.*'utitns passages in the boundary molecuies enter the agfregate througir the applopriate -interaCt and firraliy result in the ugg..gule, of th"e rvali and in the o.rt"riiru?".ia1 material with which the aggr.egate ir formed lBahadu| ct. al. l96J). All such abiogcnic morpholoqical structu|es ma). not. be. ahle to sho\v tne nronerties of biolos';ai order tut those which ha.ppened to be in the environment
order. oi such i"""T"".*,.^f;;"Ji";.ould show the prope'tiei'of biological rvhich were con'tiil,i';i{;;;;i"';;rii.i;; thoie whicl' d"pende'd on such nlaterialsprocess-'contin'ued ;;;;;i;;;t;ii"g formed in the mixturi-sa,y b^v lh.otochemical soon aftcr the suppiy of in.i. fi"iig a.fiuity uo.d th" rest ceased tireir fun-ction-sand ^the Bahadur et' a-1. 1967)'
up (Ba.hadur 1967 u.r" obr.rved in ores and rocks an'd are.reported u, ,iti.ui.'p.ri.f"rl i""ncl in sedimertta.ry rocks ar'd are described as microfossils and obseri,''ed in carbona.ceous chondritesa.ndmentioned as Orga'nised Elements' N,lanv of these are models of the earliest structures in which iife r,vas exliressed' w"s-hnirn".l necessarv'iroiecules "'""Tijr,;'J,^r"."fr**i...rtructures
Referenees B,rn.tot'e,
K., Nalure. 173, 1141 (l954) ' -
(-2) 5-B-J-602- (1964)' e"r"""", K.', lbt. bokt.,'tI7, t2i 671 (1964)' 118, Beuao,'n. K.'. 2bl. Ba',t.,
nat;t., lzt, (2\ 221-31e rls67)' lrl. ,,Jeewait',",i'n7-ir),r,rtf;, Ruttti.arainlal Bcni Pt'asad, -\llahabad Brn-ror.n. K.,
B.'il;;;;
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al., ,lbt. Bakt., 1!7, -(2) 5n584^(l?q1)',,^--, B',"ro, n K.'and C'"pia, J.L'. <!l'-81.\t" 127' ('2)' 6i5 (i9i5)' So'c".!', l2S'.7?-85 (1965)-^. sr-ro, *. K., and P;[J"',"R.-S.. i. I'tttiau tltnt' Nai'l' Aiad' Sci' India' 304' Proc' B-.,::.rrrq. K., Perti, d.K,ltJpt',1,t't, H'P',
I
.106-220 11961). I,,j,a,t J'. Al,l;. Clrtn.. ?5, 90-90 11961). 1,:.:. Br', t,,ti,i"tt)'.'J.L:.5..9.1i., Bi) J, 42, (27) 70?':71l-(1962). E.i:ri- i..'. Ir.. P.,,i,1;.fi.1 fort,ut , H' I)' and'Pande, R' S'' '4qra tlni " J' Res' . . 13, ) . 1-27 r1965). (J^qss1,^ B-re-rr,-e. K.. arLcl nur.l"n"l'or.i, s.,contbtes Rendus,!r111'ce, ?19'45.8 (.6)' 292' India,27A, Sci" Acad' Natl' l.,p,oi' B-rs-.rra. K.. i,r..d Rr;'.E;;i;.ki, jliJ ':'"it ' (lg5') ' B-rtt-$ua, K.. R"u.qa,'.avaki, S., artcl Santams\i?, !', ^Naiure'.,|| 82, 1608 R' -S'',P,erti'.9^^1T' Bes-llrn. x.. ttrr.s"ri-;"r'i, S-, -\gr-a-rva1-, K' NI'-,L', P3n-d.9, Patrika" 6' 63-117.(1963)' a,r..d Pathak. H.'D:' lt;1,rr,,n Fn'it"i.An'usandhai ear.^o;*,k., u'id ni[r^iti.':ti'i, s., ,1bt' Bakt', LL7' (2), 575-58+ (1964)'
:
.:.r-:.
-6K., and Ranga.nayaki, S., ,lbl. Bakt., ll7, (2), 567-57+ (1904). K. and Rangana.yaki, S., Vijnana Parisad Anusandhatt Patrika 9, (+),17t-182 (1e66). Banaoun; K., and.Ranganayaki,. S., Vijnana Parisad Anusandhan Patrika, 9, |7-127 (1e66). BAHaoun,-K., and Ra.nganayaki, 5., V,janona. Parisad Anusandhan Patrika. 10, (1), 5l-6r (1e67). BAuaoun, K_., and Ra-nganaya-ki, S., and Sri'astava, P., Vijnana Parisad Anusandhan PaLika, 10, (1), 5l-61, (1967). BAua.oun, F., 3f.d Ranga.na.yaki, 5., J. Brit. Interplanetary Soc., 23, (12),
BauAoun, Baneoun,
Br 3-B2e (1e70). BAuao_un, K,,, g.nd Ra.Lg1na..yaki., S., Kuma^r, Bat,t., l2O, (2\, 740-752 (1966).
A.,
ar..d Sri asta.va,
p.,
<bl.
BaHaoun, K., and Singh, Y. P. Unpublished (1971). BnnNa^r,, J, -D, _oceanography Am. Association for the Advancement of science,
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I
(1961).
Drtr.oN, L. S., Evolution,, 16, 102-l17 (1962) Flonowrrz, N. H., Proc. First Int. symposium, "The origin of Life on the Earth," Moscow, 19--24 *August 1957, Pgrg_aryon^-Press, Lond., p. 106-107 (lg5g). HALraNr, J. R. S., Rationalists Ann.,l4B (i929) Jonoo-x, P,, Ply,, 4,.,39,711 (1938; ; /. Phl's..113, 431 (1939) ; Fun.lam. radiot., 5,43 (1939)r.(. Immun Orsch. 97,330 (1940). Krrrcova, A. s., P-r'oc. First Int. Symposium, "r'he origin of Life on the Earth," * Moscow, 19,-24.Augus_t 1.957, Pergamon Pr,ess Lond., p. l16-117 (1959). L$'orr, A., A., L'euoltttion Physiologilue : etudes des pertes des fonctior,.s chez les microorganismes. Paris, 1943. Muol1_nr, P.., and RuorN, D. O., Cument Topics in Biaenergehcs, g, .
157 (1e70). I_.,Troiskhozhdenie
Izd., Moskovakii. Rabochii, Moscow, ^lhizni, j'Tle_Oligin- of _!,ift", The lvlacmillarr C-ompan1,, New york, (1938). Oe-tarx, A._f .,- "L.W ^, ILs J\rature, Origin and De:elopmertt''', Academic' press, Ner'v York (1963). P;rvtovsxanra, T. E. and.Pa.synsliii, A. G.. Proc. First,I-nt. Symposium, ,,The Origin of Life on the Earth", \4oscow l9-24, August, 1957, Pergamon Press, Lond"on pp.15l-1J7. Prnro, N. W., New Biol., 16,41 (1954). Ptnre,_ ry. W., Proc. First Int. Symposium, "The Origin of Life on the Earth,,, Moscotv, 19-21 $uguqt 1957, _Pergamon-, Press, Lond., 76-83 (tgsg). __ ILaNcaN,rvaxr,-S,, Rg.i1,a. ,!., and Ba.hadur, K., J. Brit. Interplarrct..ry soc., 25, (5), 279-286 (t972). surnNovA, A. _Y-.., Proc. Proc. rirst Int. symposium, "The origin of Life on the ^ Earth", l9-2+ August, 1957, PergamorL Pr.e55,-Lond., i84-t85 (lg5g) . StnrNne-N, .G,, Lemmon, R. M., and Ca.lvin, NI., proc. Natl. Acad. Sci., 32, 27 OpaRrN,
^A.
(192+),
(I
e64).
SrnrnneN, Q., Kenyon, D_.
E: and- Ca.lr,in, N{., J\fature, 206, 707 (1965) . G., Lemm_on, R. V., and Ca.lvin, M'., ,Science, 147: lS7+'1tOO51 . s'rnrNuex, G_., and Kenyon, D. H., "Biochemicil predeitination", Mccraw-tIil Book Comparty, Nerv York (1969). SrnrNr,rRw,
L I
A[icrograplt. "Aro.
I
: The
particles shor,vingbounda.ry wall ancl internal strtictures (x 2000 and negative magnified).
-A,Iicrograplz ..Aro. 2 : A big on. the left side ha-s
particle
a bud comirrg out on the left side (x 2000 and negative magnified) .
:r:1.,i
Micrograph .ltfo.
3 : Particles showing
tu/o generations of budding. The boun_ da"ry rvall is clearly seen (x 2000 negative magnified).
)'ficrograph l{0.4 : Abig pa-rticle shorvin"g irr.tricate intcrn:il stiuctures (x 2000 and negative magnified).
l,[icrcgral:lz "4"0.
5:
Particles
seen-
under phase contrast microsco- c. A big particle in the lo.,ver left side has its boun-dary r.vall and iniernal sti'r-rctures cleariy exposerl (x 1500 n-egative
F.
i,!
mag^n-ified).
,4,
i/
'
Micrac-raph, i\!0. 6 : Particles shoru:ir..g budding (x 2000 nega"trve ma"gnified) .
Micrograph J,[0. 7 : T]rree particles showing in ternal structures, boundary wall and buds (x 2000 negative magnifie d). L
Micrograplt"Aro. B : A particle shor,ving internal structure,
bour..darv 1'a-ll and bud ( x 2000 rr.egative magnified).
MicrograltlrNo.9:Two particles. The boundary wali
of the left particle is broken below. Internal structure and ffbudding seen (x 2000 negative ma-gnified).
hlicrograph iVo. l0 .' A parlicle r,vith broken boundarv wall sholviir.g internal stryleture ancl distjdoe8 boundary wali
(x 2000 negative
.lli.crogra.plt "nfo. I
I
magrr.ifiecl).
.' Particles under
phasecontra.st microscope. N,Iicrograph t;rken orr. colclured film. Boundarl' r,r,'all
and bud clea-rly observed (x
2000
negative nr;r.gnified)
A,Iicrograflz JVo. shor,ving
the
12 .'
N,ficrograph bor:nclat'y wall of a broken
particle like the shell of an egg rvhose content has gone out (x 2000 negative magnified)
Minograflt No. 13 .. Particles shor,vins boundary r,vall and internal sttucture
ii
(x 2000 negative magnified).
Microqraph
.l\rto.
14 .' A geng
ralg vier,v of the (x 2000).
particles